Background: Syk is a key mediator of signaling pathways downstream of several platelet surface receptors including GPVI/FcRc collagen receptor, the C-type lectin receptor CLEC-2, and integrin aIIbb3. A recent study identified the novel small molecule R406 as a selective inhibitor of Syk. Objectives: The present study evaluates the role of Syk in human platelets using the novel inhibitor R406. Methods: Agonist-induced GPVI and CLEC-2 signaling were assessed using aggregometry, immunoprecipitation and western blotting to determine the effects of R406 on platelet activation. Results: We demonstrate R406 to be a powerful inhibitor of Syk in human platelets. R406 abrogated shape change and aggregation induced by activation of GPVI and CLEC-2, and reduced platelet spreading on fibrinogen. The inhibitory effect of R406 was associated with inhibition of tyrosine phosphorylation of signaling proteins that lay downstream of Syk for all three receptors, including PLCc2.Strikingly, R406 markedly inhibited tyrosine phosphorylation of CLEC-2 and Syk downstream of CLEC-2 activation,whereas phosphorylation of Syk downstream of GPVI and integrin aIIbb3 was unaffected. Conclusions: The inhibitory effect of R406 provides direct evidence of a role for Syk in GPVI, CLEC-2 and integrin aIIbb3 signaling in human platelets. Further, the results demonstrate a critical role for Syk in mediating tyrosine phosphorylation of CLEC-2,suggesting a novel model in which both Src and Syk kinases mediate tyrosine phosphorylation of the C-type lectin receptor leading to platelet activation.